Legionella pneumophila (L. pneumophila) resides in aquatic habitats as an intracellular parasite of protozoa and when transmitted in aerosols to susceptible humans, produces an acute pneumonia known as Legionnaires'disease. Transmission of this disease is always one-way - from the environment to humans, and despite 30 years of research, an explanation for why this disease is not spread from person to person has not been offered. We have documented a developmental cycle in cell based infection models in which vegetative bacteria differentiate into cyst-like metabolically dormant forms that are resilient and highly infectious. These cysts are resistant to the effects of antibiotics, detergents and other toxic agents and can persist in water sources for extended periods where they present a health hazard. To determine if these cysts are responsible for transmission of Legionnaires'disease we propose the following aims: 1) To develop infection models in protozoa and in tap water to identify environmental conditions that promote cyst morphogenesis with sub aims of (i) tracking morphogenesis by light and electron microscopy;(i) by reporter gene fusions;(iii) pulse chase radiolabeling and proteome profiling and (iv)to assess cyst forms for resilience to detergents, biocides, and antibiotics;and infectiousness for amoebae and A/J mouse macrophages. 2) Assessment of virulence of cysts in an A/J mouse aerosol challenge model;and 3) To characterize the cyst like forms arising from protozoa,water and HeLa cells by (i) 2D gel proteome profiling for unique proteins, (ii)cyst fractionation studies to identify proteins of the outer membrane, periplasm and cytoplasmic membrane and (iii) use a mutation based approach to study developmentally regulated genes and regulatory factors. We anticipate that continued study of the planktonic cysts will contribute significant new knowledge to our understanding of the biology, pathogenesis, persistence and transmission of Legionnaires'disease. Relevance to human health. Legionnaires'disease is a rapidly growing health risk world wide, and especially for the elderly and immunocompromised. The knowledge gained from these studies should lead to improved detection and control measures that reduce the risk to humans. These studies should also provide insights relevant to other opportunistic environmental pathogens including oxiella, Burkholderia and species of Mycobacterium.